Test of the virulence and live-vaccine efficacy of auxotrophic and galE derivatives of Salmonella choleraesuis.

Aromatic compound-dependent (aro) derivatives of three mouse-virulent strains of Salmonella choleraesuis (Salmonella cholerae-suis) were constructed and shown to be nonvirulent for mice (intraperitoneal [i.p.] 50% lethal dose [LD50], greater than 5 X 10(6) CFU). A pur derivative, and a thy derivative, each of a different virulent parent, remained moderately virulent (i.p. LD50S for BALB/c mice, ca. 10(5) and 5 X 10(4) CFU, respectively). Tested as live vaccines i.p., the aro strains were ineffective in salmonella-susceptible BALB/c and C57BL/6 mice but were somewhat effective in salmonella-resistant CBA/J mice and in outbred CD-1 mice. The pur and thy strains were effective as live vaccines in BALB/c mice when given in sublethal doses. Two previously isolated nonvirulent galE derivatives of S. choleraesuis (i.p. LD50 in BALB/c mice, greater than 10(6) CFU) were also ineffective as live vaccines in BALB/c and C57BL/6 mice. The main antigenic difference between S. choleraesuis (O-6,7) and S. typhimurium (O-4,12) is in O-antigen character, thought to largely determine the specificity of protection in salmonellosis. Paired, nearly isogenic O-6,7 and O-4,12 derivatives were constructed from an aro S. typhimurium strain of proven efficacy as a live vaccine. Used as live vaccines, the O-4,12 member protected BALB/c mice against challenge with virulent S. typhimurium, whereas the O-6,7 member did not protect against virulent S. choleraesuis. However, BALB/c mice vaccinated with the O-6,7 member and mice vaccinated with an aro S. choleraesuis strain were protected against challenge with a moderately virulent (LD50, 5 X 10(4) CFU) O-6,7 derivative of an S. typhimurium strain.     

Relevance of inoculation route to virulence of three Salmonella spp. strains in mice.

The virulence of three Salmonella species strains was compared by the i.p. and i.v. routes in BALB/c mice. Salmonella choleraesuis, SL2824 (serogroup C1, O-6,7), was more virulent by the i.v. than i.p. route. A strain of S. typhimurium, SL1260 (serogroup B; O-1,4,12) was more virulent i.p. than i.v. while another strain, SL3201 (O-4,5,12) was equally virulent i.p. or i.v. The LD50 of each strain by either route correlated with the number of bacteria in the liver and spleen on day one after inoculation and thus seems determined mainly by initial bactericidal mechanisms. The rate of bacterial growth in the liver and spleen was independent of inoculation route but differed between the three strains. Salmonella choleraesuis multiplied faster than either strain of S. typhimurium. Non-virulent aromatic-dependent (aro) derivatives of these strains were tested, instead of their virulent ancestors, for survival within peritoneal macrophages in vitro. Salmonella choleraesuis SL 2824 aro and S. typhimurium SL1260 aro were much more readily killed intracellularly than S. typhimurium SL3201 aro. The data indicate that the survival and multiplication of different Salmonella serotypes or strains in vivo may depend on different critical properties or mechanisms to overcome host defenses.     

Effect of enterobacterial common antigen on mouse virulence of Salmonella typhimurium.

A series of nearly isogenic O4-12, and O-6,7 Salmonella typhimurium strains differing in regard to the enterobacterial common antigen (ECA) were constructed by conjugation. When tested in intraperitoneal infection of mice, the O-4,12 strains containing ECA were more virulent than their O-4,12 sister recombinants without ECA (P = less than 0.001). The same difference could be shown with ECA-positive and ECA-negative S. typhimurium derivatives, whose O antigens were of the group C type (O-6,7). The ECA-positive and ECA-negative O-4,12 strains did not differ in their growth rates in broth or clearance rates in vivo.     

Mice vaccinated with a non-virulent, aromatic-dependent mutant of Salmonella choleraesuis die from challenge with its virulent parent but survive challenge with Salmonella typhimurium

BALB/c mice given a live vaccine of an aroA mutant of Salmonella choleraesuis by intraperitoneal (i.p.) injection were not protected against i.p. challenge with its virulent parental strain but were protected against i.p. challenge with either of two virulent strains of Salmonella typhimurium (O [1], 4, [5], 12). Vaccination with a live vaccine of S. typhimurium aroA protected against challenge with S. typhimurium but not with S. choleraesuis. Intraperitoneal administration of either aroA strain evoked high levels of serum antibody against the homologous lipopolysacharide (LPS) as determined by an enzyme immunoassay. Sera from vaccinated mice also reacted with heterologous LPS but at dilutions at least seven-fold lower than homologous endpoint titres. The vaccination schedule employed with either live-vaccine strain conferred an equal degree of resistance to challenge with Listeria monocytogenes. After mixed infection of mice with equal numbers of virulent S. typhimurium and S. choleraesuis by the i.p. route, the former was isolated in numbers at least 50,000 times greater than the latter from the liver and spleen between days 1 and 5. When mice were vaccinated i.p. with S. choleraesuis aroA, L. monocytogenes or P. multocida before mixed infection, neither serotype showed more than a slight predominance in the liver and spleen during the same period. Thus, in relative terms, vaccination with S. choleraesuis aroA or inoculation with unrelated bacteria suppressed the growth of virulent S. typhimurium in mice but allowed virulent S. choleraesuis to multiply. These results clearly show that S. choleraesuis 38(1) can multiply to kill immunised BALB/c mice.     

O antigen as virulence factor in mouse typhoid: effect of B-cell suppression.

Immunosuppression by cyclophosphamide was used to make mice incapable of B-lymphocyte responses; they could not make an antibody response to NIP-Ficoll. These mice, as well as untreated mice, were challenged intraperitoneally with graded doses of isogenic O-4,12 or O-6,7 Salmonella typhimurium derivatives. The 50% lethal dose of the O-6,7 strains was 35- to 70-fold higher than that of the O-4,12 strains, both in the normal and the immunosuppressed animals, although the latter were approximately 1,000-fold more susceptible to the infection by either challenge organism. We conclude that the O-antigen-dependent difference in the mouse virulence of these sister strains is not mediated through differences in their capacity to evoke B-lymphocyte-mediated immune responses.     

Lysogenization of Salmonella choleraesuis by phage 14 increases average length of O-antigen chains, serum resistance and intraperitoneal mouse virulence.

Three clones from a strain of Salmonella choleraesuis (serogroup C1) were lysogenized with phage 14 (P14) which converts the O-antigen of serogroup C1 salmonellae from O-6,7 to O-6,7,14. The lysogens were compared with their parental non-lysogenic clones with respect to the following properties: average length of O-antigen polysaccharide chains, sensitivity to normal human serum, and mouse-virulence. SDS-polyacrylamide gel electrophoresis of lipopolysaccharides extracted from these bacteria showed that samples from lysogens consisted mainly of long-chained molecules whereas those from non-lysogens contained mainly short-chained molecules. The O-antigen polysaccharide from a lysogen was estimated by chemical analysis to be six times as long as that from a non-lysogen. Lysogens were serum-resistant whereas non-lysogens were serum-sensitive. About 10 times more colony forming units of a lysogen than of a non-lysogen were recovered from the livers and spleens of mice on day 1 and 3 after intraperitoneal inoculation of equal doses. By comparison with S. choleraesuis, lysogenization of S. typhimurium with phage P22 or phage A4 did not affect the chain-length distribution of O-antigen polysaccharide. Our data suggest that phage 14-coded determinants increase efficiency of O-antigen biosynthesis in S. choleraesuis leading to increase in average length of O-polysaccharide chains. Increased serum resistance and mouse virulence are logical consequences of increase in average length of O-polysaccharide chains and represent phage-conferred selective advantage not previously described in Salmonella.     

Vaccination route, infectivity and thioglycollate broth administration: effects on live vaccine efficacy of auxotrophic derivatives of Salmonella choleraesuis

An aromatic-dependent, therefore non-virulent, derivative of a mouse-virulent strain of Salmonella choleraesuis previously shown not to be effective as a live vaccine when given intraperitoneally (i.p.) to Itys mice, was administered to BALB/c mice. Two doses given i.p. or by feeding did not protect against i.p. or oral challenge with 50 to 5000 LD50 of the virulent ancestor strain. By contrast two doses given intravenously (i.v.) gave almost complete protection against i.p. or oral challenge with 500 LD50 and some protection against larger doses. The number of live bacteria (cfu) in the liver and spleen 24 h after administration of the live vaccine was less than 1% of the number inoculated i.p., but c. 25% of the number injected i.v. The number of cfu in the gut 24 h after oral vaccine administration was only c 10(-5) of the number fed. Administration of thioglycollate broth i.p. 5 days before i.p. vaccination increased recovery of live vaccine cfu in the liver and spleen and its protective efficacy. In each case the live vaccine did not multiply extensively in vivo. We have previously shown that a purine- and a thymine-requiring derivative of S. choleraesuis were each considerably attenuated but unlike the aro derivative were effective as i.p. live vaccines in mice. Doses of these strains (c. 10(4) cfu) found protective were administered i.p. to BALB/c mice. Each strain multiplied extensively in the liver and spleen to c. 10(7) cfu by day 6. All these results are in agreement with a correlation of protective efficacy of a live vaccine with the persistence of a large number of the vaccine bacteria in the liver and spleen for several days.     

Salmonella choleraesuis strains deficient in O antigen remain fully virulent for mice by parenteral inoculation but are avirulent by oral administration.

O-antigen-deficient derivatives of two mouse-virulent strains of Salmonella choleraesuis (serogroup C1; O-6,7) were constructed by transduction of a long deletion of the rfb operon. Strains SN36 and SN57 were derived from the smooth ancestor SL2824, while SN37 was derived from the smooth ancestor SL2840. These rfb deletion derivatives (rfb strains) had typical bacteriophage sensitivity patterns of "rough" Salmonella strains and were at least 200,000 times more sensitive to serum than their smooth ancestors. Lipopolysaccharides (LPS) extracted from them consisted only of two low-molecular-weight bands and lacked the ladderlike pattern of bands seen in the LPS of their smooth ancestors. The LPS from the rfb strains did not react in an enzyme immunoassay with any of three monoclonal antibodies directed against different epitopes of the O-6,7 antigen but reacted well with at least one of three monoclonal antibodies specific for core epitopes. The data were consistent with inability of these strains to synthesize O-specific chains and showed that the LPS extracted from SN57 was of chemotype Ra and that from SN36 was of chemotype Rb1, while that of SN37 consisted of a mixture of the two chemotypes. The virulence of these strains was tested by various routes in BALB/c mice. All three O-antigen-deficient derivatives were about as virulent as their "smooth" ancestors by the intraperitoneal and intravenous routes (50% lethal dose, 20 to 700 bacteria) but, unlike their ancestors, were avirulent by the oral route (50% lethal dose, greater than or equal to 5 x 10(9) bacteria). This suggests that the major role of smooth LPS in the mouse virulence of S. choleraesuis is to facilitate survival in the gastrointestinal tract and eventual entry into deeper tissues.     

Immunity conferred by Aro- Salmonella live vaccines

The specificity of protection conferred by Aro- salmonellae was studied in BALB/c mice challenged 3 months after intravenous (i.v.) vaccination, more than 1 month after the vaccine had been cleared. Oral challenge showed better protection than i.v. challenge. Salmonella typhimurium aroA SL3261 conferred very good protection against wild-type S. typhimurium C5 (over 10,000 x LD50). Cross protection experiments were performed using S. typhimurium, S. enteritidis and S. dublin for vaccination and challenge, including variants of S. typhimurium and S. enteritidis of similar virulence differing in the main LPS antigen (O-4 or O-9). Salmonella typhimurium aroA conferred solid protection against S. typhimurium (O-4), but no protection against wild-type S. enteritidis (O-9). However challenge with LPS variant strains showed that although protection was generally better to strains of the homologous LPS type, specificity of protection was determined more by the parent strain background (S. typhimurium or S. enteritidis) of the challenge than by O-factors 4 or 9, suggesting that other antigens are involved. The nature of the protective antigen(s) in this model is unclear, but it does not appear to be the main O-specific antigen. A S. enteritidis Se795 aroA vaccine gave good protection against wild-type S. enteritidis Se795 2 weeks after vaccination, but much less at 3 months (approximately 10-200 x LD50), although the persistence of the S. enteritidis aroA vaccine in the liver and spleen was similar to that of the S. typhimurium vaccine, and the wild-type Se795 challenge strain was of similar virulence to S. typhimurium C5. A S. dublin aroA vaccine conferred similar protection against wild-type S. dublin (approximately 300 x LD50).     

Rapid generation of specific protective immunity to Francisella tularensis.

Mice inoculated either subcutaneously (s.c.) or intradermally (i.d.) with a sublethal dose of Francisella tularensis LVS are immune to a lethal intraperitoneal (i.p.) or intravenous (i.v.) challenge of LVS. Here, we show that this immunity developed quite rapidly: mice given a sublethal dose of live LVS s.c. or i.d. (but not i.v.) withstood lethal i.p., i.v., or i.d. challenge as early as 2 days after the initial inoculation, despite the presence of bacterial burdens already in tissues. The magnitude of this early protection was quite impressive. The i.p. 50% lethal dose (LD50) in naive C3H/HeN mice was only 2 bacteria, while the i.p. LD50 in mice given 10(4) LVS i.d. 3 days previously was 3 x 10(6) bacteria. Similarly, the i.v. LD50 in C3H/HeN mice shifted from 3 x 10(2) in naive mice to 5 x 10(6) in primed mice within 3 days after i.d. LVS infection. Comparable changes in the i.p. and i.v. LD50 were observed in C57BL/6J mice. This rapid generation of protective immunity was specific for LVS, in that mice given a sublethal i.d. inoculation of LVS did not survive a lethal challenge with either Salmonella typhimurium W118 or Escherichia coli O118 BORT at any time, nor could mice given sublethal doses of S. typhimurium, E. coli, or Mycobacterium bovis BCG survive lethal doses of LVS. Although an increase in the mean time to death from S. typhimurium infection was noted when mice were given a sublethal i.d. dose of LVS 4 to 14 days earlier, no overall increase in protection or change in the S. typhimurium LD50 was observed. Thus, sublethal infection with LVS at skin sites induced rapid and specific protective immunity.     

Effect of the quality of the lipopolysaccharide on mouse virulence of Salmonella enteritidis.

The cell wall O antigen is known to affect mouse virulence of Salmonella. We have shown earlier that S. typhimurium expressing somatic antigen 9,12 is less virulent than its 4,12 sister transductants in mice after intraperitoneal inoculation, suggesting that the 4,12-type O antigen is connected with high virulence in mice. In this report we show, in accord with this suggestion, that when the naturally occurring O-9,12 S. enteritidis is made O-4,12 by transduction, its virulence is increased. The difference between O-9,12 and O-4,12 sister transductants is highly significant, with P less than 0.001.     

Salmonella choleraesuis and Salmonella typhimurium associated with liver cells after intravenous inoculation of rats are localized mainly in Kupffer cells and multiply intracellularly.

Male Sprague-Dawley rats were inoculated intravenously with Salmonella choleraesuis or Salmonella typhimurium and used over 3 consecutive days to produce highly enriched (greater than 95% homogenous) preparations of Kupffer and mononuclear cells (KC), liver endothelial cells (LEC), and hepatocytes. The methods involved collagenase perfusion of the liver in situ, differential centrifugation of liver cells over a Percoll gradient, and selective attachment of the cells to plastic or to culture dishes coated with collagen. The different cell preparations were then assayed for the number and location, intracellular or extracellular, of associated viable bacteria. Most of the viable bacteria recovered were associated with KC and were mainly intracellular. The intracellular bacteria in KC from rats infected with either bacterial strain increased about 20- to 50-fold over 2 days. Some of the bacteria associated with LEC and in some experiments with hepatocytes also survived treatment with gentamicin and increased in number with time. Intracellular bacteria were readily visualized in KC by light microscopy and transmission electron microscopy. On rare occasions, bacteria were seen within LEC from rats infected with S. choleraesuis but not from those infected with S. typhimurium. Microcolonies of S. typhimurium but not of S. choleraesuis were occasionally found on the surface of some LEC. Bacteria were not seen within or on the surface of hepatocytes by transmission or scanning electron microscopy. The integration of microscopic and viability data suggested that most intracellular S. choleraesuis organisms in KC had been killed whereas most intracellular S. typhimurium organisms were viable.     

Role of phagocytosis in mouse virulence of Salmonella typhimurium recombinants with O antigen 6,7 or 4,12.

The quality of lipopolysaccharide has previously been shown to influence the mouse virulence of Salmonella so that strains with O antigen 4,12 were more virulent than their O-9,12 sister strains. Immunosuppression did not alter this O-antigen-dependent difference in virulence. I have now constructed smooth O-4,12 and O-6,7 sister hybrid strains of Salmonella typhimurium. No other phenotypic differences were found between these strains; they were all "common antigen" positive. In intraperitoneal infection, the O-4,12 strains were more mouse virulent than their O-6,7 sisters. The difference in virulence correlated with a difference in clearance rates; the O-6,7 hybrids were removed from the blood more rapidly than their O-4,12 sisters. No natural bactericidal antibodies were found in the sera of the mice.     

Systemic and enteric colonization of pigs by a hilA signature-tagged mutant of Salmonella choleraesuis

Although host adapted to pigs, Salmonella enterica serovar Choleraesuis (S. choleraesuis) can induce a virulent foodborne salmonellosis in humans. To directly investigate virulence factors of S. choleraesuis, we extended the functional genomics approach of signature-tagged mutagenesis to S. choleraesuis and pigs. When a test pool of 45 randomly signature-tagged null mutants was inoculated orally and intraperitoneally in pigs, one of the mutants that failed to colonize by either route was tagged in hilA. In the broad host range serovar S. typhimurium, hilA regulates invasion genes in the first pathogenicity island and is required for enteric but not systemic infections in mice experimentally infected with S. typhimurium. The pool of tagged S. choleraesuis null mutants was a complex mix inoculated in pigs. When pigs were challenged with an equal mixture of the hilA mutant and wild type bacteria, the hilA mutant was at a competitive disadvantaged (attenuated) in pigs inoculated orally but not in intraperitoneally inoculated pigs. Our data support that hilA in S. choleraesuis infections of pigs has a role in enteric but not systemic infections similar to that of S. typhimurium in the murine model of human typhoid fever. The role of hilA may be conserved across Salmonella serovars and host species.     

Host specificity of Salmonella infection in chickens and mice is expressed in vivo primarily at the level of the reticuloendothelial system.

By experimental infection, host-specific Salmonella serotypes were shown to demonstrate specificities for chickens, mice, and other laboratory animals. Following oral inoculation, four strains of Salmonella gallinarum and two S. pullorum strains, isolated from diseased poultry, were more virulent for chickens than for mice. By contrast, four strains each of S. choleraesuis and S. dublin, isolated from diseased pigs and cattle, respectively, were more virulent for mice than for chickens. These results were also reflected in the degree of virulence expressed after parenteral inoculation. In addition, S. choleraesuis, but not other serotypes, killed rats, guinea pigs, and rabbits. S. typhimurium strains varied widely in their virulence, and some strains were virulent for both mice and chickens. Four other serotypes isolated from poultry or human food poisoning cases and a nonpathogenic Escherichia coli strain were much less virulent for both experimental host species. Most of the host-specific Salmonella serotypes studied were able to colonize the distal alimentary tract and invade the tissues in both mice and chickens to various degrees. There was, however, a greater difference in the ability to survive and multiply in the visceral organs, particularly the spleen and the liver, once invasion had occurred which correlated with the virulence for the host species involved.     

Oral immunization of mice and swine with an attenuated Salmonella choleraesuis [delta cya-12 delta(crp-cdt)19] mutant containing a recombinant plasmid.

Salmonella choleraesuis chi 3781, an attenuated [delta cya-12 delta(crp-cdt)19] mutant, was electroporated with the plasmid pBA31-R7, which codes for the expression of a 31-kDa protein from Brucella abortus (BCSP31). Recombinant S. choleraesuis chi 3781 stably maintained the pBA31-R7 plasmid and continued to express the cloned protein following recovery of the organism from orally inoculated animals. Unlike previous studies using S. typhimurium chi 4064(pBA31-R7), S. choleraesuis chi 3781(pBA31-R7) was able to colonize both the gut mucosa and deep tissues of both BALB/cByJ mice and crossbred swine. Orally inoculated mice developed serum antibodies to both the cloned 31-kDa protein (rBCSP31) and to S. choleraesuis chi 3781 endotoxin. These mice also developed a local intestinal antibody response to Salmonella endotoxin but not to rBCSP31. Similarly, mice inoculated with recombinant S. choleraesuis chi 3781 did not develop a delayed-type hypersensitivity (DTH) footpad response following injection with rBCSP31; however, these mice did respond to S. choleraesuis chi 3781 soluble antigen. Conversely, orally inoculated swine did not develop significant serum or intestinal antibody responses to cloned protein or Salmonella endotoxin, but DTH responses to both cloned protein and S. choleraesuis chi 3781 soluble antigen were strongly positive. The cell-mediated nature of these DTH responses was confirmed by histological examination. Results suggest that S. choleraesuis chi 3781 may be a suitable choice for further studies of vaccine efficacy in swine, especially for diseases which require cell-mediated immunity for resolution.     

Protection and immune responses induced by attenuated Salmonella typhimurium UK-1 strains

We previously reported that Salmonella typhimurium SR-11 mutants with deletion mutations in the genes encoding adenylate cyclase (cya) and the cAMP receptor protein (crp) are avirulent and protective in mice. Salmonella typhimurium UK-1 is highly virulent for chicks (oral LD50 of 3x10(3) CFU) and mice (oral LD50 of 8.5x10(3) CFU) and is capable of lethal infections in pigs, calves and horses. We postulated that attenuated derivatives of this lethal strain would probably induce a higher level of protective immunity than achieved with attenuated derivatives of less virulent S. typhimurium strains such as SR11. To test this hypothesis, we have constructed S. typhimurium UK-1 Deltacya-12Deltacrp-11 mutant strain chi3985 and its virulence plasmid cured derivative chi4095 to investigate their avirulence and immunogenicity in mice. We found that the mutants are avirulent and able to induce protective immune responses in BALB/c mice. These mutant strains retained wild-type ability to colonize the gut associated lymphoid tissue but reach and persist in spleen and liver at a significantly lower level than the wild-type parent strain. Mice survived oral infection with >1x10(9) CFU of chi3985 (the equivalent to 10(5) 50% lethal doses of wild-type S. typhimurium UK-1) and were fully protected against challenge with 10(5)times the LD50 of the wild-type parent. Immunized mice developed a high level of serum IgG titre to Salmonella LPS and delayed-type hypersensitivity (DTH) response to S. typhimurium outer membrane proteins. Compared to the virulence plasmid-containing strain chi3985, the virulence plasmid cured DeltacyaDeltacrp mutant strain chi4095 was more attenuated and less protective, as some mice immunized with chi4095 died when challenged with the wild-type UK-1 strain. This work demonstrates that S. typhimurium UK-1 Deltacrp Deltacya mutant strain may be a potential live vaccine to induce protective immunity against Salmonella infection or to deliver foreign antigens to the immune system.     

Some galE mutants of Salmonella choleraesuis retain virulence.

galE mutants were isolated from three mouse-virulent strains of Salmonella choleraesuis (of group C1, O antigen 6,7) by selection for resistance to 2-deoxygalactose. The galE derivative of strain 381 comprised two components: galactose sensitive, thought to be the original mutant; and galactose resistant, presumably by a second mutation reducing galK or galT function or both. The galactose-sensitive component had an intraperitoneal 50% lethal dose for BALB/c mice of ca. 4 X 10(6) CFU, whereas the galactose-resistant component was about as virulent as its gal+ parent, with a 50% lethal dose of ca. 100 CFU. The galE mutant of strain 110 was somewhat sensitive to galactose, as shown by retardation of growth; its 50% lethal dose, ca. 500 CFU, was not much greater than the ca. 200 CFU value for its parent. The galE mutant of strain 117 showed the same partial sensitivity to galactose as strain 110 galE, but was nonvirulent (50% lethal dose of ca. 10(6) CFU versus ca. 400 CFU for its parent). Growth on galactose-supplemented medium restored the smooth phenotype, as indicated by phage sensitivity to three of the four galE strains, but only partially so for the strain 117 galE mutant. The retention of parental virulence by galE mutants of S. choleraesuis which are galactose resistant or somewhat galactose sensitive contrasts with the greatly reduced virulence of galactose-resistant galE mutants of Salmonella typhimurium and Salmonella typhi; this difference may result from the absence of galactose from the O repeat unit in the lipopolysaccharide of group C1 salmonellae.     

Lipopolysaccharide alteration mediated by the virulence plasmid of Salmonella

Lipopolysaccharide (LPS) of Salmonella dublin, S. enteritidis, S. typhimurium, S. choleraesuis and their derivative strains was analysed to investigate the correlation between LPS and virulence plasmid of Salmonella. All wild-type strains had smooth type LPS, i.e. LPS with long O-specific polysaccharide. The virulence plasmid-cured strain of S. dublin, C524, exhibited a shorter O-specific chain than its parent strain, 5240. No distinct ladder bands were observed at the high molecular weight region on the SDS-PAGE gel for C524 LPS. By chemical analysis the number of O-repeating unit of C524 LPS was shown to be approximately one. The chain length of O-specific polysaccharide was restored by reintroduction of the virulence plasmid. The alteration of LPS by curing and reintroduction of the virulence plasmid was not observed when other wild-type strains of S. dublin were used. In the case of S. enteritidis, S. typhimurium, and S. choleraesuis, alteration of neither chemical composition nor electrophoretical profile of LPS was detected by curing and reintroduction of the virulence plasmids. Those results suggest that certain factor for regulation of the chain length of O-specific polysaccharide is encoded on the virulence plasmid of S. dublin.     

Listeria monocytogenes, but not Salmonella typhimurium, elicits a CD18-independent mechanism of neutrophil extravasation into the murine peritoneal cavity.

This study shows that extravasation of neutrophils into the peritoneal cavities of mice in response to intraperitoneal (i.p.) inoculation of wild-type Listeria monocytogenes requires the participation of leukocyte adhesion molecules that are different from those involved in neutrophil recruitment in response to i.p. inoculation of Salmonella typhimurium. In the case of S. typhimurium, extensive neutrophil influx could be essentially abolished by treating mice with either anti-CD11b or anti-CD18 monoclonal antibodies, whereas the same monoclonal antibodies failed to prevent neutrophil accumulation in the peritoneal cavity in response to inoculation of L. monocytogenes. On the other hand, i.p. inoculation of a listeriolysin-negative strain of L. monocytogenes induced a CD11b-dependent neutrophil influx. The possibility that wild-type L. monocytogenes, by virtue of its ability to inhabit the cytosol of the cells it infects, induces the expression of endothelial cell adhesion molecules in the microvasculature of the peritoneal cavity to which neutrophils adhere via leukocyte adhesion molecules distinct from beta-2 integrins is discussed.